Cancer incidence is progressively increasing in parallel with an
increase in the rate of cancer survivors with the help of advanced
treatment modalities. By the year 2010, it is estimated that one in
every 250 persons will have survived a childhood malignancy. The
increased rates of survival bring about complications related to
reproductive health. Cytotoxic treatments due to chemo- and radiotherapy
or bone marrow transplantation suppress or irreversibly harm not only
female ovarian reserve but also male testicular sperm production. In
this review, cryopreservation of gametes and gonads with fertility
preservation options and indications prior to cancer treatments are
discussed. (Turk J Hematol 2009; 26: 106-13)

Cancer incidence is gradually increasing while deaths caused by
malignant diseases decrease. When all female cancers are considered,
despite an increase in the cancer incidence by 0.3% annually from 1987
to 1999, the death rates for all cancers combined decreased by 0.6%
annually from 1992 to 1999 as a result of improvements in current
treatment modalities including surgical techniques, radiation therapy,
multi-agent chemotherapy, and hematopoietic stem cell transplantation
(HSCT) [1, 2]. By 2010, one in every 250 persons is estimated to have
survived childhood malignancies. Thus, more patients survive cancer
every year but face the challenging long-term side effects, especially
related to their reproductive system. The risk of ovarian failure may
increase up to nine-fold in female cancer survivors receiving
cyclophosphamide-based combination chemotherapy [3, 4], and ovarian
failure is almost inevitable in patients undergoing preconditioning with
chemoradiation before HSCT [5]. It is also reported that an ovarian
radiation dose of more than 6 Gy usually results in permanent
infertility [6]. Chemo-/radiotherapy sequelae may impair the quality of
the pregnancy as well, with an increased risk of early pregnancy loss,
premature labor, and low birth weight, even if the patients were not
sterilized after treatments [7-9].

The introduction of assisted reproduction and its current worldwide
utilization has resulted in the development of successful
cryopreservation techniques for surplus embryos. The techniques created
for embryo cryopreservation have further been applied to the
unfertilized mature and immature human oocyte [10, 11] and ovarian
tissue [12-17]. Currently, embryo cryopreservation is the preferred
method to preserve future fertility because of reasonable post-thaw
survival, implantation, and delivery rates, if the patient has a
life-partner or access to a sperm donation program. However, oocyte or
ovarian cryopreservation can be the solitary option for young or
unmarried female patients. Pre-pubertal children, on the grounds of
ethical concerns regarding ovulation induction and oocyte retrieval, and
women who cannot delay cancer treatment for the 2-4 week period
necessary to perform ovulation induction, are not candidates for embryo
cryopreservation.

Numerous non-neoplastic diseases are also treated with cytotoxic
chemotherapy and radiation. In some of them, chemotherapy or radiation
therapy is also used in extreme doses to ablate the preexisting bone
marrow in HSCT. The indications for HSCT that extend beyond cancer now
include some autoimmune diseases unresponsive to immunosuppressive
therapy, diseases associated with genetically abnormal stem cells
(hemoglobinopathies and enzyme deficiency disorders), and those
associated with the deficiency of bone marrow stem cell products
[18-23]. In this article, the current indications and techniques of
fertility preservation will be reviewed from the gynecologic point of
view, which mainly focuses on embryo, oocyte and ovarian
cryopreservation.

Chemotherapy-and radiotherapy-associated gonadal damage

Multiagent chemotherapy constitutes the basis of the modern cancer
treatment. Ovaries, which are stocked with irreplaceable follicles, are
extremely sensitive to most cytotoxic drugs [23, 24]. The end result of
the chemotherapy can range from damage to steroid-producing cells and/or
oocytes of developing ovarian follicles (granulosa and theca cells),
which can cause temporary amenorrhea, to apoptotic death of primordial
follicles, which results in premature ovarian failure (POF).
Ultrastructurally, ovarian exposure to chemotherapeutics is associated
with marked follicle loss [25]. Factors that can potentially modify the
risk of chemotherapy-induced ovarian failure are summarized in Table 1.

Some chemotherapeutic agents are more commonly associated with
permanent and irreversible gonadal damage, such as cyclophosphamide,
chlorambucil, melphalan, busulfan, nitrogen mustard, procarbazine,
ifosfamide, and thiotepa [23, 26-30]. Among the moderately gonadotoxic
agents are cisplatin and adriamycin, while bleomycin, actinomycin D,
vincristine, methotrexate, and 5-fluorouracil are associated with mild
or no gonadotoxicity (Table 2). Although there is limited evidence,
paclitaxel may also be gonadotoxic, but this remains to be verified
[31]. In Table 2, we classify the gonadal risk of commonly used
chemotherapeutic agents after a comprehensive literature search [32].

Cyclophosphamide is the most recognized agent to cause damage to
oocytes and granulosa cells. In a recent mouse study,
cyclophosphamide-induced follicular damage occurred in a dose-dependent
manner, even at low doses of 20 mg/kg [33]. Relative risk of POF was
reported to be between 4 and 9.3 in patients receiving cyclophosphamide
[34, 35]. During the last 10-15 years before the onset of menopause,
primordial follicle loss is accelerated, which is reflected by a
constant decrease in inhibin B levels and increase in
follicle-stimulating hormone (FSH) levels. As a consequence, a smaller
number of follicles that are more prone to cell division errors begin to
grow each cycle, until menopause occurs, when the number of follicles
falls below 1.000. Because of this, older women with a low primordial
follicle pool have a higher risk of developing ovarian failure compared
with young women with higher primordial follicle numbers. Consistent
with this biological fact, earlier studies demonstrated that a
cumulative cyclophosphamide dose of 5.2 g caused amenorrhea in women in
their forties, 9.3 g in women in their thirties, and 20.4 g in women in
their twenties [27].

Hematopoietic stem cell transplantation and the risk of ovarian
failure

There has been a dramatic increase in the survival of childhood
cancer patients in recent years as a result of HSCT. On the contrary,
high-dose chemotherapy used for conditioning before HSCT is extremely
gonadotoxic, as has been consistently demonstrated in the previous
studies. In the acute ovarian failure of childhood cancer survivor study
[36] that included 3309 childhood cancer survivors, exposure to more
than 1000 cGy ovarian radiation, age and treatment with cyclophosphamide
or procarbazine were found as independent risk factors for development
of POF in a multivariable logistic regression model. An important
finding from the previous studies is that despite timely menarche, FSH
concentrations show a tendency to rise to menopausal levels in children
exposed to high-dose chemotherapy during the pre-pubertal period. This
highlights the fact that occurrence of timely menarche does not
guarantee preserved ovarian function. Brachet et al. [37] found that
seven of 10 children with sickle cell disease receiving busulfan (14 or
16 mg/kg) and cyclophosphamide (200 mg/kg) as preconditioning before
HSCT developed POF. In the remaining three who had spontaneous puberty,
serum FSH levels were very high at the time of puberty and slowly
normalized thereafter. It is important to underline that three girls
with ovarian function recovery differed from the seven others by the
lower busulphan dose of the conditioning regimen they received (14
rather than 16 mg/kg). In a survey including 2819 childhood cancer
survivors, Sklar et al. [38] demonstrated that children who receive
chemotherapy are at an extremely high risk for POF. In that study, the
authors followed a cohort of children who were diagnosed with a
malignancy before the age of 21 and were menstruating for at least five
years afterward. The patients in the study group were compared with
their 1065 siblings. The median age at diagnosis was 7 (range, 0-20) and
the median age at study was 29 (range, 18-50). The risk of developing
ovarian failure was found 13.2-fold increased (range, 3.26-53.51) in
those exposed to chemotherapy compared with their siblings. Although
rare, resumption of menstruation years after the diagnosis of POF has
also been reported [39].

How to assess post-chemotherapy gonadal function?

Most of the long-term follow-up studies assessing post-chemotherapy
ovarian function rely on menstruation as the only surrogate marker. Even
though irregular menstruation or amenorrhea is highly likely to occur
during the chemotherapy, even lasting for a considerable period after
completion of the chemotherapy, many patients return to a
pre-chemotherapy menstrual pattern. Hormonal reversal of a
hypergonadotropic state that commonly occurs during the courses of
chemotherapy to a normo-gonadotropic state may also be expected [40].
However, these women will always have a high risk of developing
premature menopause during their later reproductive life. The fact that
ovulation may occur despite loss of half of the follicular pool in
rodents indicates that indirect assessment of ovarian reserve is an
unreliable tool [23]. Ovarian reserve diminishes when FSH levels on the
third day of the menstrual cycle are more than 12 IU/ml or estradiol is
more than 75 pg/ ml, whereas ovarian failure is diagnosed when FSH is
found as more than 40 mIU/ml in two measurements regardless of menstrual
bleeding. Anti-Mullerian hormone (AMH) has recently been suggested as
the most reliable marker of ovarian reserve [41]. In normal ovulating
women, serum AMH levels are relatively constant during the menstrual
cycle, serum concentrations of which show a rapid decline after 37
years. Anderson et al. [42] showed that compared with estradiol and FSH,
AMH showed a more rapid and sustained change after chemotherapy.
Moreover, the decrease in AMH occurred without a significant decrease in
inhibin-B or increase in FSH concentrations. The severity and rapidity
of the decrease in AMH concentrations compared with the partial decline
in inhibin-B concentrations might reflect primordial and preantral
follicles as the primary site of toxicity. This supports the observation
that, even though there may be no clinical signs of ovarian failure,
there is always damage to follicular reserve in proportion to the
cumulative dose of chemotherapeutic agents that might not be detectable
with routinely used laboratory tests. It is important to note that AMH
is not influenced by confounding factors such as oral contraceptive use,
day of menstrual cycle, or pregnancy. In a study assessing
postchemotherapy ovarian function [43], despite the fact that all eight
breast cancer study patients resumed menstruation after chemotherapy,
three had irregular menstrual cycles, and five had undetectable
inhibin-B levels or FSH values more than 50 IU/ml, suggesting some
degree of impairment in ovarian reserve. Another study [44] found that,
compared with FSH and inhibin-B, AMH constitutes the most sensitive
predictor of ovarian reserve in women treated with chemotherapy for
Hodgkin's lymphoma. Furthermore, most women who reported one or
more pregnancies had normal AMH levels for age at the time of the study.
Similarly, it has been demonstrated that in breast cancer patients, AMH
levels declined despite continued menstrual activity [45] and ovarian
reserve markers were altered in those who seemingly had normal
menstruation postchemotherapy [46]. Giuseppe et al. [47] assessed FSH,
luteinizing hormone (LH), AMH, inhibin-B, and antral follicle count
(AFC) and suggested the combination of AFC and AMH as having the best
predictive value for ovarian reserve with a high sensitivity (83%) and
specificity (88%) in patients treated with chemotherapy for
Hodgkin's lymphoma. In 25 patients with hematological malignancies,
serum AMH concentrations were measured before and after cancer therapy
and compared with normoovulatory controls. Despite having menstrual
cycles and despite some patients conceiving spontaneously after
chemotherapy, AMH levels and AFC were decreased, showing some degree of
ovarian damage [48].

Radiotherapy

Ionizing radiation is a well-recognized cause of ovarian damage and
permanent infertility. Gonadal damage occurs not only by direct exposure
to radiation such as in the case of pelvic or low abdominal irradiation,
but scattering of radiation may also cause considerable damage even if
gonads are outside the radiation field. Radiation causes a dose-related
reduction in the primordial follicle pool [49]. The human oocyte is
extremely sensitive to radiation, and irradiation at ovarian dose >6
Gy usually causes irreversible ovarian failure [6]. Wallace et al. [50,
51] demonstrated that <4 Gy is enough to destroy half of the oocyte
population (LDL50 <4 Gy); however, very recently, using a revised
mathematical model, the same authors suggested that the LDL50 of the
oocytes was <2 Gy. Age at the time of exposure to radiotherapy,
extent and type of radiation therapy (e.g. abdominal, pelvic external
beam irradiation, intracavitary brachytherapy) and fractionation
schedule are important prognostic indicators for development of ovarian
failure [52-56]. In mice, radiation-induced chromosome damage in the
oocytes was more evident in older compared with younger animals [54]. In
general, irradiation is more toxic when given in single dose compared to
fractionated doses. Stillman et al. [57] investigated the risk of
ovarian failure among 182 long-term survivors of childhood cancers
receiving abdominal radiotherapy. The mean follow-up was 16.4 years.
Ovarian failure occurred in 68% of the patients when both ovaries were
in the irradiation field, and in 14% of the patients when both ovaries
were at the edge of the treatment field. None of the 122 children
developed ovarian failure when one or both ovaries were outside the
abdominal treatment field. In another study, failure in pubertal
development or premature menopause was observed in 37 of 38 patients who
received external abdominal irradiation during childhood for
intraabdominal tumors in doses ranging from 20 to 30 Gy [58]. Sanders et
al. [59] reported the probability of ovarian failure in patients
receiving cyclophosphamide and total body irradiation for HSCT as 1.00
at one year. Failure in pubertal development may be the first sign of
ovarian failure in these patients who received radiotherapy during
childhood.

Indications for fertility preservation

As a result of improvements in cancer treatment and in the ability
to detect tumors in their early stages by well-established screening
programs for some cancers, life expectancy has strikingly increased.
Furthermore, a cure is now possible for many childhood and adult
cancers. Notably, cure rates approximate 90% in certain childhood
cancers. A beneficial effect of cytotoxic treatment in various
non-malignant diseases has also been repeatedly demonstrated. The idea
of cryopreserving ovarian tissue is based on the finding that the
ovarian cortex harbors primordial follicles that are more resistant to
cryo-injury than are mature oocytes. Although the clinical indications
for ovarian tissue cryopreservation are almost identical to those for
the oocyte, there are fewer logistical restrictions in offering this
technique. Despite the limited data on successful pregnancy rates,
ovarian tissue cryopreservation has broader applications and, in theory,
a greater fertility potential than oocyte cryopreservation because of
the far larger number of oocytes preserved. Extending indications for
ovarian tissue cryopreservation are listed below. A detailed list of
indications is presented in Table 3 [60].

Cancers in children

Adult and childhood cancers are the most common indication for
fertility preservation. Even though cancer is still the second leading
cause of death in children, there has been remarkable improvement in the
cure rates of many childhood cancers over the last three decades [1].
Among the most common cancers encountered during childhood are leukemia,
Hodgkin's and non-Hodgkin's lymphomas, tumors of the central
nervous system, soft tissue sarcomas, and renal tumors [61-66]. Acute
lymphoblastic leukemia is the most common childhood cancer, with more
than 2.100 new cases and 2,000 long-term survivors each year [66, 67].
The five-year survival rate for all childhood cancers is approximately
80%, with a higher percentage for lymphomas (94%) and Wilms' tumor
(91%) [66-69]. Although many children survive cancer because of improved
treatment modalities, they are certainly not immune to the gonadotoxic
effects of various cancer treatments. Ovarian tissue cryopreservation
may be the only acceptable method for any pre-pubertal or pre-menarchal
female patients receiving chemotherapy, pelvic radiotherapy, HSCT, or
oophorectomy for benign disease or prophylaxis [23]. The greatest
benefit from the procedure is expected in children, since they have the
highest number of primordial follicles [16]. With ovarian tissue
freezing, no ovarian stimulation is needed; therefore, time restrictions
for cancer therapy are fewer, and there is no risk of stimulating
estrogen-sensitive cancer following ovarian stimulation [23].
Additionally, it avoids ethical concerns regarding ovarian stimulation
and oocyte retrieval in children.

Cancers in adults

The death rates from cancer in women have fallen, despite increased
incidence during the 1990s. Approximately 8% of these cancers occur in
reproductive aged women under the age of 40 years. Breast cancer, the
most common cancer in women during the reproductive years, afflicted
approximately 216,000 women in the United States in 2004 [66]. The
five-year survival rate in breast cancer now approaches 90%. Most of the
patients with breast cancer are subjected to cyclophosphamide-based
gonadotoxic chemotherapy. In breast cancer, fortunately, unlike other
malignant diseases, there is approximately a six-week hiatus between the
initial surgery and chemotherapy. These patients may resort to assisted
reproductive technologies during this time period. However, in theory,
conventional ovarian stimulation protocols are thought to affect the
growth of breast cancer as a result of supraphysiological estrogen
concentrations. Novel stimulation protocols with tamoxifen and aromatase
inhibitors are suggested as safer protocols in these patients. In
addition, since occult ovarian metastasis is extremely rare, with the
exception of stage IV disease and lobular carcinoma, these patients may
resort to ovarian tissue cryopreservation [23,70]. Cancer of the cervix
is a serious health problem afflicting 500.000 women worldwide each
year, with almost half of them under the age of 35 [71]. Patients with
advanced stage disease and those with early stage disease who are found
to have high risk factors receive pelvic or pelvic/paraaortic radiation
therapy. Squamous cell cancer of the cervix, which is the most often
encountered subtype, rarely metastasizes to the ovaries, whereas this
may occur at a rate as high as 12% for adenocarcinoma and adenosquamous
carcinoma. Ovarian transposition might be performed; however, success
rates vary greatly because of damage to the vasculature during the
procedure. Ovulation induction might be risky since there is risk of
bleeding from the fragile cervix during oocyte retrieval. Ovarian tissue
can be removed in selected patients for cryopreservation during primary
cancer surgery. Another group of patients that are potential candidates
for ovarian cryopreservation are those carrying BRCA I and II mutations.
Despite the fact that the risk of peritoneal cancer cannot be totally
eliminated in BRCA-positive patients, prophylactic oophorectomy is
suggested as soon as childbearing is completed or by the age 35-40 years
to decrease the risk of ovarian and breast cancer [72,73]. Cortical
pieces of ovarian tissue in those with a desire for fertility can be
frozen for future use.

Autoimmune diseases

Autoimmune diseases can also affect women of reproductive age.
There have been an increasing number of reports regarding the use of
cytotoxic treatment, especially with cyclophosphamide, in autoimmune
diseases, including systemic lupus erythematosus, steroid- resistant
glomerulonephritis, Behcet's disease, inflammatory bowel diseases,
and pemphigus vulgaris [74-78]. Pieces of ovarian tissue may be
harvested for possible future use in these patients in order to retain
fertility.

Experimental approaches

When the risk of ovarian involvement with cancer cells is high,
some other experimental options may be considered. It has been possible
to isolate primordial follicles from human ovarian tissue, but there has
been no success in growing them in vitro to get a healthy offspring
[79]. Early stage preantral follicles can only be grown for brief
periods of time in three-dimensional culture systems. Another potential
approach is xenografting human ovarian tissue in immunodeficient mice,
where human follicles can be grown to antral stages and ovulated.
However, the applicability of xenografting in the clinical setting has
not been determined due to the risk of trans-species viral infections
[80, 81]. The mechanism of age-related as well as chemo- or
radiotherapy-induced loss in the ovarian germ cell population is
proposed to be mediated by programmed cell death, i.e. apoptosis.
Sphingosine-1phosphate (S1P), a bioactive sphingolipid metabolite, is an
important lipid mediator and has many actions both inside and outside
the cell. It was demonstrated that wild-type mice treated with S1P
resisted both developmental and cancer therapy-induced apoptosis.
Radiation-induced oocyte loss could be completely prevented by S1P
therapy in wild-type mice, and no genomic damage in mice pretreated with
S1P before receiving ionizing radiation could be demonstrated [82].
Another experimental approach has become a current issue with a report
by Silber et al. [83], in which a transplantation of ovarian cortical
tissue took place between 24-year-old monozygotic twins, one of whom
suffered POF. The question arises of whether allogeneic ovarian
transplantation is possible in the future for females with ovarian
dysfunction following cancer therapies.

Conclusion

Fertility preservation requires a multimodality approach. Depending
on a patient's age, the type of cancer treated, time constraints,
availability of a partner, and whether there is ovarian involvement, a
different procedure may be needed for each cancer survivor facing
treatment-related infertility. Physicians should take a comprehensive
approach in counseling their patients regarding fertility preservation
procedures.